Document 3J2D4jrL6DyqVxdmJkmGJGrZ6

OIFN V3ITRCOHMMPIACNYR'OSBIOCLOOMGBIOCUANLD TM-U3T6A5G1ENICITY ASSAYS mite' . Final Report ac-- February 1985 sy: MTia_cchrKlotetteendlOkGseansetroO i:csMbiScerpsoebreLt moeTnoygTE g Had-lic b Fhvart 5t . RiccioT, gAsi sTotao nt DIFector Prepared for: HG3eednieCcroaamllpaODnfyufpiacreasentM Center Se. Paul, Ho 53144 Attention: TWoixlilciomlogC.y MeGomatck Specialist [CT] Mciicrsoibioanl sGenteotrivcosimmDneeprartsmiernteer TTozn 3. etd, eDicretct4s) Tortontegs Liborsanrs 0.6 Mw, VL.iteA scSicetnmcmeesr,DiVviicsetoPnrestdent =e ae on SACS 000073 SUMMARY SRI International examined 34 Company's Compound T-3651 for mutagenic activity in the standard Ames Salmonella/microscme assay with strains TAL535, TALS37, TA1538, TA9, and TA100 of the bacterium Salmonella typhimurium. Compound T-3651 vas also screened for recombinogenic activity io the yeast Saccharomyces cerevisiae D3 assay. All assays were perforned in the presence and absence of a rat-liver metabolic activation system. Compound T-3651 was reproducibly mommutagenic and nonrecombinogenic when tested according to these procedures. 11 000074 CONTENTS INTR++sO ensnD ssnsU snsC nsesT enseI nannO ennnN nnnn. nes 1 METHODS..evvvvnnnnnnnnnnsssssssssssansnnsannnnnens 5 RESULTS AND DISCUSSION. .eeeeevvvevnnnnnnnnananns 12 TABLES PPE | Table 2eevrereeeneeneennnninniiniiinneenns 14 Table uuuuiuiunniiiiiienenenneinnenaiiaaees 15 Table buuennnnnnnnnniinaeeaaaaeaeanenannnnns 16 111 000075 INTRODUCTION SRI International examined 34 Company's Compound T-3651 for mutagenicity in the standard Ames Salmonella/microscme assay with strains TALSIS, TALSI7, TAISIS, TA9, and TAL0D of the bacterium Salmonella typhimurium. Compound T-3651 was also tested for reccabinogenic activity in the yeast Saccharomyces cerevisiae D3 assay. An Aroclor 125stinulated, rat-liver homogenate metabolic activation system was included 1n the assay procedures to provide metabolic steps that the microorganisss either are incapable of conducting or do not carry out under the assay conditions. The assay procedure vith S. typhisuriwm has proven to be 80 to 90% reliable in detecting carcinogens as mutagens, and it has bout the same reliability in identifying chemicals that are not carcinogenic. The assay procedure with S. cerevisiae is about 60% reliable in detecting carcino gens as agents that increase mitotic recombination. However, because the assay systems do not always provide 100% correlation with carcinogenicity investigations in animals, neither a positive tor a negative response conclusively proves that a chemical 1s carcinogenic or noncarcinogenic to man. Evaluation of experimental results from the Salmonella assay consists of comparing the number of histidine-independent colonies on the treated agar' plates with the number observed on the control plates. Because all the plated Salmonella indicator organisms undergo a few cell divisions in the presence of the test chemical, the test 1s semiquantitative in nature. The plate test procedure does mot permit quantitative determination of the number of cells surviving the chemical treatment. It is the demonstration of a mutagenic dose-response relationship that Ls important in establiching mutagentetty. 1 000076 The test chemicals are assayed at several dose levels within a nom toxic dose range---vith the exception of the highest dose level, which sometimes exhibits toxicity. Toxicity is evidenced by several phenomena: clearing of the background bacterial lawn growth, formation of pinpoint colonies consisting of surviving cells, and a decrease in the number of revertant colonies below the spontaneous background. A chemical is considered a mutagen in the Salmonella assay if it elicits a reproducible, dose-related increase in the number of histidine Tevertants per plate in one or more tester strains. The yeast Saccharouyces cerevisiae D3 is a eukaryotic microorganism capable of detecting mitotic recombination, as expressed through a mutation leading to a defective enzyme in the adenine-metabolizing pathway, resulting in a red-pigmented colony. In this assay, the yeast cells are exposed to several concentrations of the test chemical, usually ranging from a concentration that results in no killing to ome that causes 50% killing. Any concentration that induces 90% killing is considered toxic. When the aumber of genetically altered colonies per milliliter (yield) amd the ratio of altered colonies to survivors (frequency) from the treated cells are unequivocally larger than those of the solvent-treated controls, we com clude that the exposure of the cells to the compound induces mitotic recom bination. If this event is dose-related, the observation is termed a positive response. 2 000077 MATERIALS Test Articles - Names: T-3651 - Date Received: 28 November 1984 - Description: Amber liquid (foms at high concentrations) ~ Storage Conditions: Room temperature - Special Testing Conditions: Mone - Stability: Assured by Sponsor Indicator Organisms - Species: Salmonella typhimurium LT2; Saccharomyces cerevisiae - Strains: tyTApLhSi3nSu,rTfAuL;SI7D,3 TfAoLrS3SS., cTeArSeSv,isiaaned TAL0D for S- - Source: Dr. the Bruce Ames, Salmonella; DUrn.iveF.rsiK.tyZiofmmeCramlainfno,rniWa., GeBremrakneyl,ey,forfor the yeast Metabolic Activation A~r2o6c.l5ormg1/2a5l4-ipnrdoutceeidn, rat liver S-9; SRI Batch Fi; Negative (Diluent) Control Material Sterile vater Positive Control Chemicals 9-Animoacridine, CAS Manufacturer: Pfaltz No. and 90-45-9 Bauer, Stamford, CT 3 000078 2-Anthramine, CAS Mo. 613-13-8 Manufacturer: Sigas Chemical Co., St. Louis, MO 2-Nitrofluorene, CAS No. 607-57-8 Manufacturer: Aldrich Chemical Co., Milwaukee, WI Sodium Azide, CAS Fo. 26628-22-8 Manufacturer: Difco Laboratories, Detroit, MI 1,2:3,4-Diepoxybutane, CAS No. 1464-535 Manufacturer: Pfaltz and Bauer, Stamford, CT Sterigmatocystin, CAS No. 10048-13-2 Manufacturer: Aldrich Chemical Co., Milwaukee, WI Counters Used =~ New Brunswick Scientific Counter, Model Cl11, SRI BioTran II Automated Nos. 0030 6126 00 and Colony 0012 3318 00 - New Brunswick Scientific C110, SRI Nos. 0013 0788 Bactronic Colony Counter, 00 and 0030 1471 00 Model 4 000079 METHODS Salmonella typhisuriw Strains TALS3S, TALS7, TALS38, TAS, end TALOO The Salmonella typhtauriwm strains ued at SRI are all histidine awrotzophs by virtue of mutations in the histidine operon. When these hiscidine-depeadent cells are grown on ataizal mediwm agar plates com taining a trace of histidine, only those cells that revert to histidine tadependence (his*) are able to form colonies. The small mount of his tidine allovs all the plated bacteria to undergo a few divisions; in many cases, this growth is essential for mutagenesis to occur. The his* revercants are easily visible as colonies against the slight background growth. The spontaneous mucation frequency of each strain is relatively constant, but when a autagen is added to the agar, the mutation frequency is increased, usually in a dose-related manner. We obtained our S. typhisurium strains fram Dr. Bruce Ames of the University of California at Berkeley. In addition to having mutations in the histidine operon, all the indicator strains have a mutation (rf) that leads to a defective lipopolysaccharide coat; they also have a deletion that covers genes involved in the synthesis of the vitamin biotin (bio) and fn the repair of Wtraviolet (w)-induced DNA damage (wvrB). The cfs mutation makes the strains more permeable to many large molecules, thereby increasing the mutagenic effect of these molecules. ThewrBmutation renders the bacteria wable to use the accurate excision repair mechanism to remove certatn chemically or physically induced DNA lesions and thereby eahances the strains' sensitivity to same mutagenic agents. Strain TASS 1s reverted to his* by many mutagens that cause base-pair substitutions. Strain TALOD 1s derived fram TALSIS by the {ntroduction of the resistance trmsfer factor, plasaid pRAIOL. This plasmid is believed to cause m tacrease in error-prone DNA repair that leads to many more mutations for a given dose of most mutagens. In addition, plasmid pRYLOL confers resis 5 000050 tasce to the meibiotic mplcillin, which is a convenient marker to detect the presence of the plasaid fn the cell. The presence of this plasmid also makes strain TAL0O sensitive to some frameshift mutagens [e.g., ICR-L91, benzo(a)pyrene, aflatoxin By, and 7,12-dnethylbenz(s)anthracene]. Strains TALS}? md TALSIS are reverted by meny frameshift muagens. Strain TA% is derived from TALS by the addition of the plasaid pRHIOL, which makes it more sensitive to some mutagenic agents. AL tndicator strains are kept frozen in nutrient broth supplesented with 10% sterile glycerol at ~80C in l-al aliquots containing about 10 cells. New frozen stock cultures are made every three months from single colony 1solates that have been checked for their gemtypic characteristics (his, rfa, wrB, blo) and for the presence of the plasmid. For each experiment, the frozen l-ul cell cultures are allowed to thaw at roca temperature before inoculation in 50 al of glucose mininal liquid medium supplemented with an excess of biotin and histidine. The cultures are grown at 37C, unshaken for 4 hours, then gently shaken (100 rpm) for 11 to 14 hours. All strains are genetically analyzed whenever experiments are perforaed. Aroclor 1254-Stiaulated Metabolic Activation System Some carcinogenic chemicals (e.g., of the aromatic mine type or the polycyclic hydrocarbon type) are inactive unless they are metabolized to active forus. In animals and aan, an enzyme system in the liver or other organs (e.g., lung or kidney) is capable of metabolizing a large number of these chemicals to carcinogens. Some of these intermediate metabolites are very potent mutagens in the S. typhimurium test. Ames has described the liver metabolic activation system that we use. In brief, adult male Sprague-Davley rats (200 to 250 g) are given a single 500-ng/kg {ntraperitoneal injection of Aroclor 1254 (a mixture of polychlorinated biphenyls). This treatment enhances the synthesis of enzymes fmvolved in the metabolic conversion of chemicals. Four days after the injection, the mimals' food 16 removed but drinking water is provadilidbieewdm. On the fifth day, the rats are killed ad the liver homogenate is prepared as follovs. 6 000081 The livers are removed aseptically and placed in a preweighed, ster1le glass beaker. The organ weight is deternined, and all subsequent operations are conducted in a ice bath. The livers are washed vith an equal volume of cold, sterile 0.15 KCl, minced vith sterile surgical scissors in three volumes of 0.15 M KCl (3 ml/g of wet organ), and homo genized vith a Potter-Elvehjem apparatus. The homogenate is centrifuged for 10 minutes at 9000 x g, and the supernatant, referred to as the 5-9 fraction, is quickly frozen on dry ice and stored at -80C. The metabolic activation mixture for each experiment consists of, for 50 al: 5.0 ml of $9 fraction 1.0 ml of gCl, (0.4 ) and KCl (1.65 M) 0.25 wl of glucose-6-phosphate (1 M) 2.0 ml of NADP (0.1 ) 25.0 ml of sodium phosphate buffer (0.2 M, pH 7.4) 16.75 ml of sterile HO. The amount of $9 fraction delivered to each plate is 50 ul. Plate Incorporation Assay Prior to testing, the test article is serially diluted from m ini- tial stock. In some cases, a preliminary experiment is conducted to find a suitable dose range for testing. The article is usually tested over a aint of six dose levels, the highest nontoxic dose level being 10 mg/ plate wless solubility, mutagenicity, or toxicity dictates a lower upper limit. When extracts are made, various undiluted aliquots are tested, usually over a dose range of 5 to 100 or 200 yl/plate. When liquids are tested, occasionally the sample is not diluted and various aliquots ere used. All assays are repeated at least once on a separate day. The plate incorporation assay is performed in the following way. To a sterile 13 x 100-m test tube placed in a 43C heating block we add: 7 00005 (1) 2u.n0d00.a0l5 oafx0.hi6s7tiadgianrecontatning 0.6% NaCl, 0.05 4 biotin, ((23)) 00..0055 mmll ooff aindsioclauttoironoorfganthiesmstes(taboaurtcic1l0e8 bacteria) (4) 0.50 m1 of metabolic activation nixcure (if appropriate). This mixture 1s stirred gently and then poured on plates containirg about 25 nl of nintaal glucose sgar. After the top agar has sec, the plates are Incubated for 48 hours at 37C. The mumber of his" revertant colontes is comted wing a BoTran II automated colony counter when possible. When accurate counts cant be obtained (e.g. because of precipitate), the Plates are counted sanually ueteg mm electric probe colony comter+ trolsCoanrceurrruemntwitshteerivleirtyy,expneergiaateinvte. (soSlcveernitl/idciyluceonntt)r,olsandinpclousdietivpelatcionng= owt separately steps (3) and @). For negative controls, we use steps (a1r)t,icl(e2,),1f(4a),ppraonpdri0a.t0e5. mlFoorf tphoesitsiovlevecnotn/tdriolluse,ntweusetdestfoeracthhebatcetsetrial culture with the following mutagens using steps (1), (2), (3), and (4): oSoptttm sazide for the base-palr subscicucton mucants TALS3S S-Antnoscridine for the frameshife mutant TALS] 2-Nitrofluorene for the fraseshife mutants TALSIS and TASS a2e-tAanbtohlriacmtnaeccifvoartiaolnl. tester strains, in the presence of Criterta for Interpretation Posteive. A test arcicle fo considered a mutagen shen it produces a reproducible, dose-related increase in the number of revertants fa one or more strains. This fncreass must occur for at least three done levels: Negative, A test arcicle 1s considered a somutagen vhen no doserelated faccesse in the mmber of revertants 1s observed fn at least two independent experiaents. The maxtaus done level tested for macoric cospounds 1s 10 sg/plate (unless dictated otherwise by solubility probe Lem. Tor torte compounds, only the highest dose level tested should show evidence of toxicity. . 000083 Inconclusive. When a test article cannot be identified clearly a5 a mutagen or momutagen in the standard plate assay, the results are classified as tncosclustve. Saccharcuyces cerevisiseD3 The yeast S. cerevisise D3 is a diploid microorganisa heterozygous for a mutation leading to a defective enyzme in the adenine-metabolizing pathway. When grown on medium containing adenine, cells homozygous for this mutation produce a red pigment. These homozygous mutants can be generated from the heterozygotes by mitotic recoabination. The frequency of this recoubinational event may be increased by fmcubacing the organtsas with various carcinogenic or reccmbinogenic agents. The recambinogenic activity of a compound or 1ts metabolite is deternined from the number of red-pignented colonies appearing on test plates. A stock culture of S. cerevisiae is stored at 4C. For each experi ment, broth conta fning 0.05% gSO,, 0.15%KH R0,, 0.451 (NH,)SO0,, 0.35% peptone, 0.5% yeast extract, and 2% dextrose is inoculated with a loopful of the stock culture and incubated overnight at 30C, with shaking. Theinvitro yeast mitotic recombination asay in suspension is conducted as follows. The overnight culture is centrifuged and the cells are resuspended at a concentration of 108 cells/al in 67 wf phosphate buffer (pH 7.4). To a sterile test tube are added: 1.0 ul of the resuspended culture 0.5 al of either the metabolic activation mixture or buffer 0.2 ul of the test cheateal 0.3 al of buffer. Several doses of the test chemical are tested (up to SZ w/v or v/v) in each experiment, ant appropriate solvent/diluent controls are included. 1,2:3,4-Diepoxybucane without metabolic activation ad sterigaatocystin vith activation are used as positive controls. The suspension mixture is tncubated at 30C for 4 hours on a roller drum. The sample ie then diluted serially in stertle physiologic saline, and 0.2 ul of the 1075 and 1073 dilutions is spread on plates containing 9 ccon84 the same ingredients as the broth plus 2.0% agar; five plates are spread with the 1073 dilution and three plates are spread with the 1075 dilution. The plates are incubated for 3 days at 30C, followed by at least 1 day at 4C to enhance the development of the red pignent indicative of adenine-deficient homozygosity. Plates containing the 1073 dilution are scanned with a dissecting microscope at 10x magnification, and the number of mitotic recoabinants (red colonies or red sectors) is recorded. The surviving fraction of organisms 1s deternined from the total number of colonies appearing on the plates of the 1075 dilution. Criteria for Interpretation Positive. A positive response in this assay is indicated by a dose related increase of more than threefold in the absolute number of mitotic recombinants per milliliter and in the relative number of mitotic recombinants per 105 survivors. Negative. When no reproducible recoubinogenic sctivity is obtained in any of the assays performed, the test results are considered to be negative. Incomlusive. When a test article cannot be identified clearly as causing a positive or a negative response, the results are classified as inconclusive. Statistical Analysts No statistical malysis was performed for my of the assays. The results of the plate incorporation assay are a tabulation of the number of colonies appearing on the plates. The results of the S. cerevisiae D3 assay are tabulated after calculating the number of mitotic recombinants per 105 survivors. ALL calculations are expressed with two significant dsgtes. 10 000085 References Aasmesf,ramB.eshNi.,ftE.autG.ageGnusr:ney,MeJt.aboAliMteisllearn,d daendrivH.atiBvaerstscohf.2-aCcaertcyilnaomgiennosUfSlAuo6r9e,ne31m2d8-3o1t3h2er(1a9r7o2m)a.tic mine carcinogens. Proc. Natl. Acad. Sci. Ammuetsa,genBs.: N.,A Ws.impE.le Duterssttons,ystE.emTcaommabsiankiin,ganldiveFr. Dh.omoLgeee.nateCsarcfoirnogaecntsivaatrieon nd bacteria for detection. Proc. Natl. Acad. Sci. USA 10, 2281-2285 asm). sayesst,em B.forN.,theF.deD.teclteieo,nanadndW.clEa.ssDiufrisctaotni.on oAfn miumtpargoevends banadctecrairaclinotgeesnts. Proc. atl. Acad. Sci.USA 10, 782-786 (1973). Agmeenss, anB.d Na.u,tagJ.ensMcCwaintnh, thaend SEa.lmYoanmealslaak/im.msaMleitahno-dsmicfroorsdoemteecmtuitnaggecniacrictiynotest. Mutat. Res.31, 347-360 (1575). Birngusttcekc,hniDq.ueJs., wiatnhd Vy.easWt.. MayEearv.ironN.ewHedaelvtehlopPmeernstpescti.n m,uta83g-e8n6ici(t1y973s)c.reen Kinierc,igaLr.etDt.e, Es.mokYeamacsoankdie,nsaatneds.B. PN.rocA.vesN.atl.DeAtceacdt.ioSncio.f mUuStAa7g1e,nic415a9c-t4i1v6i3ty EDN MgecnCsanna,s oJ.u,tagEe.nsChoiin,thE.e SYaalmsasoankeil,la/aandicrBo.scNe.e Amteesst.: DAestseacytioofn o30f0ccahrceimno icals. Proc. Natl. Acad. Sci.USA 72, 5135-5139 (1975). McCann, J., and B. N. Anes. Detection of carcinogens as mutagens in the SNaatllm.onAeclalda./miSccir.osoUSmAe 7t3e,st:90-A9s5s%ay(o1f9763)0.0 chemicals: Discussion. Proc. Mpororpteerltaymso,f Kp.lasEa.,ldaRnd46B.fAr.oDn. itSetouckletrr.avioSleegtr-epgraottieocntionfgthperopmeurttayt.or Mol. Gen. Genet. 167, 317-327 (1979). Zviitmhaernaiatnrno,usF.aciKd.,, 1a-nmdetRh.ylS-c3h-vnaiiterr.o-l-Innidturcotsioognusonfidmiinteotiacndgoetnheerconavlkeyrlsaito-n ing agents lo Saccharoayces cerevisiae. Hol. Gen. Genet. 100, 3-76 sen). un 000086 RESULTS AND DISCUSSION 3 Conpany's Compound T-3651 was screened for mutagenic activity fa the Anes Salmonella/afcroscmeinvitromutagenicity assay using the five standard strains of Salmonella Eyphimurium: TALS3S, TALS37, TALS3S, TASS, and TAL00. The assays vere perforaed in duplicate, both in the presence and abseace of a rat-liver metabolic activation system. Sterile water was used as the diluent. The microbial mutagenicity testing of this sample vas performed on 12 and 18 December 1984. Dose levels ranging from 10 to 5000 pg/plate were used for all Ames assays (Tables 1 and 2). Compound T-3651 fomed when it vas diluted vith water to form the stock solution of 100 mg/al; further dilutions vere perforaed once the fom settled. Mo dose-related increases in the number of histidine-independent revertants vere observed with the coapound in either assay. Compound T-3651 was also screened for recombioogentc activity fn the yeast Saccharcayces cerevisise D3 assay for aitotic reccabination. The assays were performed on 10 and 29 January 1985, both with and without metabolic activation (Tables 3 and 4). Sterile vater vas used as the diluent. At the high doses, T3651 fomed md appeared to react with vater. No dose-related focreases in the number of mitotic recoubinaats per 105 survivors were observed vith the test compound in either assay. In concluston, Compound T-3651 vas reproducibly mutagenic and nourecombinogenic when tested according to these procedures. 12 000087 Table 1 DN VITRO ASSAYS WITH SANOWLIA TYRUDURION courompT-3651 Expertnent Date: 12 Deceber 1984 Conpound Neesbolte CoAmdpdoedund Activation per Plate TATE M7satItSdine ReTvearstasnts eorTPlate TT Negative Control ater -+ O50 u 1B9 72 010 106 1 ooua 2w5 o39e1s39 wlee Postetve Controls z SSondtimoomschretidietne -n2kinctheraamitnees -- FlLu dk a weno as a s1 130% 1166 10427 0982 wm ue -: : 21 5 5 auso ae a0 oowm Be we 6m Compound T3651 z: 5M0 e 100 2Bo%aoL9 Bono4 9G7 1omm oaow. nm15do1 we an s19 :: - 1050000 5000 aWon 9 6 15 was asuo oa 8 10 wu 301smops l1ae 2 25 1s 120 ++ : 5100 100 Mo 90o9ruo12ess6 21s o1u5 we w3 owows ony aoimm 2 +++ 3100000 5000 VBo1s6 4oe [ARUSEECIRR ou6 o1a00 o12u ER 32 367dme l1%e ge3@ Tote 0 11m0 srscownirommps1u-3p61e TYP Ertan tutes 18 December 198 Copmd dreewcsiordsees pCeor"mhptaogiuned TTT Haotttine setvereance pesmcheate [air---- -: B #7 mhEoa aTe n01l0 wBaus mum iw RSoCotctinhetCtonetrools I, Lie mw oa _x Femi ::: :3i Bowowm o 1Ss2ahons aoNehD633 eRs R0s Cov Tae : =:B moan ow X [CF2I E BR SN RE punmoam SS: owmmeweoonmBBooahoogdEGS ooBg opdNJNI FP mBaspBaioonowmow ::.: ww oouwcw0oaoomdaoda5 nono nhd ohoR w Ranam nomoiw REE 2gg Csooeoms aolel B4OaF RBRN BREwoeow 88 Toe 3 IN Imo ASSATS OF siccmmances cmevisue 03 [re Expeciacat Date: 10 Januncy 1985 Compound Metabolic GoncPeerncceenattion Cselulrsviveirngal Survivors ReWceomrblsneants per al ReNceamobtinsgate oe 105, activation ow he io Tih sivivers' Negative Control Vater -: 6S1s 110000 `2 s6i0 Fostetve controle 5 1,2b5u3c,an-e.dtegory- Steripatocyetin o.0.m0s05 66s2 5 on. 1062 + 0.005 pd 10 0 300 Compound 1-3651 -: 00..105 : os prnam 110000 100 110 12} :- 1so0 761 10100 23 2u.s8 ++ 00..105 + 0.5 5o.s8 11000 IX] 100 "s 26s a6 ++ 1pe0} 7pss 110% s5 peast gg ALL calculations are expressed to two significant Figures. 3$8 || 1m sos x s--_c--r_am coins | petacovoen 13930sy 190 | EPR t TEar[-- oS sd i woe wwaine. nto omer : [ e aat-- n a: T CCwEmon@wn m8 a wmPoRmme <gg$ [rT gg